| 质粒名称: | v3em-Nterm-PE2max |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 207861 |
| 存储实验室: | David Liu |
|
载体骨架基本信息 |
|
| 载体名称: | p601 modified |
| 载体类型: | AAV细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | NpuC-Cterm-PE6c-dualU6-Rosa26-twinPE-attB |
| 物种来源: | Cas9 is from S. pyogenes; Tf1 RT is from the Tf1 retrotransposon |
| 插入大小: | 2775bp |
| 启动子: | Cbh |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | AGCGAGTCAGTGAGCGAG |
| 3’测序引物 | GTTTTCCCAGTCACGAC |
|
文献引用方法 |
|
| 材料和方法部分: | v3em-Cterm-PE6c-dualU6-Rosa26-twinPE-attB was a gift from David Liu (Addgene plasmid # 207861 ; http://n2t.net/addgene:207861 ; RRID:Addgene_207861) |
| 参考文献部分: | Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Doman JL, Pandey S, Neugebauer ME, An M, Davis JR, Randolph PB, McElroy A, Gao XD, Raguram A, Richter MF, Everette KA, Banskota S, Tian K, Tao YA, Tolar J, Osborn MJ, Liu DR. Cell. 2023 Aug 31;186(18):3983-4002.e26. doi: 10.1016/j.cell.2023.07.039. 10.1016/j.cell.2023.07.039 PubMed 37657419 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS v3em-Cterm-PE6c-dualU6-Rosa26-twinPE-attB 7809 bp ds-DNA circular SYN 19-SEP-2023
DEFINITION AAV genome encoding C-terminal PE6c and U6 expression cassettes for
Rosa26 twinPE pegRNA pairs.
ACCESSION .
VERSION .
KEYWORDS v3em-Cterm-PE6c-dualU6-Rosa26-twinPE-attB
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7809)
AUTHORS Doman JL, Pandey S, Neugebauer ME, An M, Davis JR, Randolph PB,
McElroy A, Gao XD, Raguram A, Richter MF, Everette KA, Banskota S,
Tian K, Tao YA, Tolar J, Osborn MJ, Liu DR
TITLE Phage-assisted evolution and protein engineering yield compact,
efficient prime editors.
JOURNAL Cell. 2023 Aug 31;186(18):3983-4002.e26. doi:
10.1016/j.cell.2023.07.039.
PUBMED 37657419
REFERENCE 2 (bases 1 to 7809)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 19, 2023 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7809
/organism="synthetic DNA construct"
/mol_type="other DNA"
rep_origin 1..589
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 490..509
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 743..760
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 877..898
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 913..943
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 951..967
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 956..978
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 975..991
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 975..991
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
repeat_region 1018..1147
/label=AAV2 ITR
enhancer 1170..1455
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
promoter 1457..1734
/label=chicken beta-actin promoter
intron 1735..1962
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
CDS 2016..2036
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3231..3251
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4731..4751
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
primer_bind 4771..4790
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
misc_RNA complement(5000..5075)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(5098..5411)
/label=U6 promoter
/note="RNA polymerase III promoter for mouse U6 snRNA (Das
et al., 1988)"
primer_bind complement(5159..5179)
/label=mU6-F
/note="Mouse U6 promoter, forward primer"
misc_RNA complement(5518..5593)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(5623..5863)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
primer_bind complement(5673..5692)
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
primer_bind complement(5843..5863)
/label=hU6-F
/note="Human U6 promoter, forward primer"
repeat_region 5887..6016
/label=AAV2 ITR
primer_bind complement(6035..6052)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(6035..6051)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6044..6066)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 6193..6648
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6280..6299)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 6490..6511
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 6674..6778
/gene="bla"
/label=AmpR promoter
CDS 6779..7639
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(6997..7016)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
ORIGIN
1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
961 ataacaattt cacacaggaa acagctatga ccatgattac gccagattta attaaggctg
1021 cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt
1081 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
1141 ggttcctgcg gcctctagat cagggtaccc gttacataac ttacggtaaa tggcccgcct
1201 ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc
1261 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
1321 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
1381 tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
1441 atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc
1501 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg
1561 gcgggggggg ggggggggcg cgcgccaggs ggggsggggs ggggsgaggg gcggggcggg
1621 gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt
1681 atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc
1741 gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
1801 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
1861 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg
1921 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca
1981 tgaaacggac agccgacgga agcgagttcg agtcaccaaa gaagaagcgg aaagtcatca
2041 agattgctac acggaaatac ctgggaaagc agaacgtgta cgacatcggc gtggagcggg
2101 atcacaactt cgccctgaag aatggcttta tcgccagcaa ttgtttcaac gaaatcggca
2161 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta
2221 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg
2281 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc
2341 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc
2401 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt
2461 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa
2521 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa
2581 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga
2641 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga
2701 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca
2761 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg
2821 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg
2881 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc
2941 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc
3001 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca
3061 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga
3121 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggt gactccggcg
3181 gaagctctgg tggcagcaag cggaccgccg acggctctga attcgagagc cctaagaaga
3241 aaagaaaggt gagcggaggc tctagcggcg gaagcatcag cagctctaag cacaccctga
3301 gccagatgaa caaggtgagc aacatcgtga aggaacccga gctgcccgac atctacaagg
3361 aatttaagga catcaccgcc gacaccaata ccgagaagct gcctaagcct attaagggcc
3421 tggaatttga agtggaactg acacaggaga actacagact gcctatccgg aactatcctc
3481 tgactccagt caagatgcag gccatgaacg acgagatcaa tcaaggcctg aaaggcggca
3541 tcatcagaga gagcaaggcc atcaacgcct gccctgttat attcgtgccc agaaaggaag
3601 gcacactgcg catggtggtc gactacaggc ccctgaacaa gtacgtgaag cccaacgtct
3661 accccctgcc actgattgaa caactgctgg ccaagatcca gggcagcacc attttcacca
3721 agctggacct gaaaagcgcc taccaccaga tcagagtgcg aaagggagat gagcacaagc
3781 tggccttccg gtgccccaga ggagtcttcg agtacctggt gatgccttac ggcatcaaga
3841 cagcccctgc ccactttcag tacttcatca acacaatcct gggcgaggcc aaggaaagcc
3901 acgtggtgtg ctacatggac gacatcctga tccattccaa gtccgagtcc gaacacgtga
3961 aacatgtgaa ggacgtgctg caaaagctga agaacgctaa tctgatcatc aaccaggcca
4021 aatgcgagtt tcaccagagc caagtgaagt tcctgggcta ccacatcagc gagaagggct
4081 taaccccatg tcaggagaac atcgacaagg tgctgcagtg gaaacagcct aaaaaccaga
4141 aggaactgag acagttcctg ggccaggtga actacctgag aaaattcatc cccaaaacca
4201 gccagttgac ccaccctctg aacaaactcc tgaaaaagga tgtcagatgg aaatggaccc
4261 ctacccagac ccaggctatc gagaatatca agcaatgtct ggtgtctcct cctgtgctga
4321 ggcacttcga cttcagcaag aagatcctgc ttgagacaga cgtttctgat gtggccgtgg
4381 gagccgtgct gagccagaag catgatgatg ataagtacta ccctgtgggc tattacagcg
4441 ctaaaatgag caaagcccag ctgaattatt ctgtgtccga caaggagatg ctggctatca
4501 tcaaaagcct ggagcactgg cggcactacc tggaatctac aatcgagccc ttcaagatcc
4561 tgaccgacca cagaaacctg attggaagaa tcacaaacga gagcgaacca gagaacaagc
4621 ggctggccag atggcagctg ttcctgcagg acttcaactt cgagatcaac tacagacctg
4681 gctcttctgg cggctcaaaa agaaccgccg acggcagcga attcgagtct cccaagaaga
4741 agaggaaagt ctaagtcgac ctttatttgt gaaatttgtg atgctattgc tttatttgta
4801 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
4861 gttcaggggg agatgtggga ggttttttaa agcaagcttg caaaaaaatt ctagttggtt
4921 taacgcgtaa ctagatagaa ccgcgctact gttcactcta aggcttgtcg acgacggcgg
4981 tctccgtcgt caggatcatg caccgactcg gtgccacttt ttcaagttga taacggacta
5041 gccttatttt aacttgctat ttctagctct aaaacctgtt agagtgaaca gtagacaaac
5101 aaggcttttc tccaagggat atttatagtc tcaaaacaca caattacttt acagttaggg
5161 tgagtttcct tttgtgctgt tttttaaaat aataatttag tatttgtatc tcttatagaa
5221 atccaagcct atcatgtaaa atgtagctag tattaaaaag aacagattat ctgtctttta
5281 tcgcacatta agcctctata gttactagga aatattatat gcaaattaac cggggcaggg
5341 gagtagccga gcttctccca caagtctgtg cgagggggcc ggcgcgggcc tagagatggc
5401 ggcgtcggat cgcgaattca aaaaaattct agttggttta acgcgtaact agatagaacc
5461 gcgaatctgc tagtatatca tgatcctgac gacggagacc gccgtcgtcg acaagccgca
5521 ccgactcggt gccacttttt caagttgata acggactagc cttattttaa cttgctattt
5581 ctagctctaa aacacggata tactagcaga ttacggtgtt tcgtcctttc cacaagatat
5641 ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa
5701 aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt
5761 gtactaatat ctttgtgttt acagtcaaat taattctaat tatctctcta acagccttgt
5821 atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcttcctgc ccgaccttgc
5881 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
5941 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
6001 gcgagcgagc gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg
6061 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
6121 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
6181 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
6241 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
6301 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
6361 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
6421 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
6481 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
6541 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
6601 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
6661 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
6721 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
6781 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
6841 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
6901 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
6961 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
7021 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
7081 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
7141 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
7201 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
7261 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
7321 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
7381 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
7441 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
7501 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
7561 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
7621 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
7681 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
7741 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
7801 aggatcttc
//
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